Method for stabilizing coenzyme and composition thereof

ABSTRACT

Disclosed is a sugar and/or a sugar alcohol as a substance for suppressing dephosphorylation reaction of a phosphorylated coenzyme. Also disclosed is a method for stabilizing a phosphorylated coenzyme which is characterized by having at least a substance for suppressing dephosphorylation reaction of the phosphorylated coenzyme coexist with the phosphorylated coenzyme.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.11/576,048, which is a national stage of International ApplicationPCT/JP2005/018424, filed Oct. 5, 2005, which claims priority to JapaneseApplication No. 2004-292710, filed Oct. 5, 2004. The disclosures ofapplication Ser. No. 11/576,048 and PCT/JP2005/018424 are incorporatedby reference herein in their entireties.

TECHNICAL FIELD

The present invention relates to a stabilization method for aphosphorylated coenzyme characterized by allowing at least a substancefor suppressing dephosphorylation reaction of the phosphorylatedcoenzyme to coexist with the phosphorylated coenzyme, a preservationmethod therefor, or a stabilized composition. The present invention isused in the field of clinical diagnosis, food evaluation, and an assayfor biogenic substances.

BACKGROUND ART

Heretofore, coenzymes have been frequently used for chemical reagentsfor biochemical clinical investigations. Coenzymes, which have beenused, include oxidation or reduction type nicotinamide adeninedinucleotide (NADH or NAD) or nicotinamide adenine dinucleotidephosphate (NADPH or NADP). However, those coenzymes have a problem withstability. The coenzyme changes to an oxidized coenzyme, NAD or NADP, bya very small amount of an enzyme included in an assay reagent, ordisintegrates ADP ribose and others, thereby resulting in a change instructure which can be used by an enzyme. Therefore, there is a problemin that such a change leads to a decrease in total amount of thecoenzyme and then lowers the sensitivity of the assay agent (Non-patentDocument 1). For solving this problem, it is known that a coenzyme ispreserved under temperature conditions as low as possible, such as underfreezer storage or cold storage and a coenzyme-containing reagent isthen freeze-dried. Alternatively, it is known that, as stabilizers forpreserving the coenzyme in a state of solution, there are used, forexample, an amine base, sodium hydroxide, a chelating agent, azide,boric acid, an alkali metal, an ammonium bicarbonate buffer, and anactive-oxygen removing substance. Any of these methods has beenconceived with the intention of preventing the coenzyme from beingdecomposed as described above to avoid a decrease in sensitivity (PatentDocument 1 and Patent Document 2).

Coenzymes are used for immunologic clinical diagnostic reagents. Forinstance, in an enzymatic immunoassay, there has been known acoenzyme-cycling method as a method of detecting an alkaline-phosphatase(ALP)-labeled antibody with a phosphorylated coenzyme, NADP or NADPH,which is used as a substrate (Patent Document 3). Detection methodsusing enzyme-cycling reagents made of NADP or NADPH, alcoholdehydrogenase, diaphorase, alcohol, and tetrazolium salt have been wellknown in the art. For example, when NADP is used as a substrate for ALP,NAD is produced from NADP by ALP and the resulting NAD is then convertedinto NADP by alcohol dehydrogenase and alcohol. Subsequently, NADHreturns to NAD by diaphorase and tetrazolium salt and the reaction ofconverting NAD to NADH then proceeds again. In this way, through thecyclic reaction between NAD and NADH, formazan dyes produced fromtetrazolium salt accumulate in a reaction solution. Thus, the amount ofthe dyes is measured to determine the activity of ALP.

Even those reagents also have problems with stability of coenzyme, likebiochemical reagents, for prolonged preservation of the reagents,coenzymes have been preserved in a free-dried state under temperatureconditions as low as possible, such as under freezer storage or coldstorage. In addition, an attempt has been made by selecting anappropriate buffer for stabilization.

In contrast, in recent years, simple assays, which are typified by assayreagents for influenza antigen and capable of obtaining assay results onthe spot in a medical office or at bedside, have been becoming popular.Any of these point-of-care (POC) test reagents requires, if it should bepreserved under cold storage or preservation of lower temperature, aspecific device, such as a refrigerator or a freezer, and thepreservation space for the reagent is also limited, so there is aproblem in that a large amount of the reagent cannot be purchased andpreserved. Therefore, a reagent, which can be preserved at roomtemperature, has been demanded. However, in these POC test reagents, forproviding a reagent using a coenzyme for practical use, which can bepreserved at room temperature, a prolonged preservation stability undersevere conditions than the conventional refrigerating preservation hasbeen already difficult as described above and now such a preservationstability should be attained under more severe conditions. Therefore,the practical use of a test reagent using a coenzyme in this field hasbeen extremely difficult.

In addition, in the field of test reagents, in order to measure a verysmall amount of a substance of interest, it is demanded to enhance thesensitivity of the measurement. ALP has been well known as a targetenzyme generally used in an enzymatic immunoassay, so a procedure ofdetecting ALP at high sensitivity has been demanded. In addition, thetechnique of enhancing the sensitivity is useful not only for microassaybut also for reduction in amount of a sample or shortening the measuringtime. Thus, this is the most desired technique in this field.

-   [Patent Document 1] JP 3470099 B-   [Patent Document 2] JP 2001-61498 A-   [Patent Document 3] JP 06-303996 A-   [Non-patent Document 1] Basic experimental method for protein and    enzyme, Nankodo Co., Ltd., page 426

DISCLOSURE OF THE INVENTION Problem to be Solved by the Invention

It is an object of the present invention to provide a stabilizationmethod for a phosphorylated coenzyme for a prolonged period particularlyat room temperature.

Means for Solving the Problem

The inventors of the present invention have concentrated on intensivestudy in consideration of the following viewpoints in particular tosolve the above-mentioned problems. As a result, the inventors of thepresent invention have finally completed the present invention byfinding out a stabilization method for a coenzyme, by which the coenzymecan be stably preserved at room temperature for a prolonged time. In anassay for ALP activity using a coenzyme, it is very difficult to attainthe practical use of a reagent that allows the assay to be stablyperformed with good reproducibility for a prolonged time. Besides, inparticular, for the practical use of a reagent for ALP activity assaywith the use of a high-sensitive coenzyme-cycling method, which can bepreserved at room temperature for a prolonged time, there is a need ofthe technique of coenzyme stabilization, which is capable of loweringnon-specific color development which increases in proportion to anincrease in preservation period. Thus, the inventors of the presentinvention have studied in consideration of limitations in knownstabilization techniques and an importance in finding of a novelstabilization method.

That is, the present invention relates to a stabilization method for aphosphorylated coenzyme, characterized by allowing at least a substancefor suppressing dephosphorylation reaction of the phosphorylatedcoenzyme to coexist with the phosphorylated coenzyme, and relates to:

(1) a stabilization method for a phosphorylated coenzyme, comprisingallowing at least a substance for suppressing dephosphorylation reactionof a phosphorylated coenzyme to coexist with a phosphorylated coenzyme

(2) a stabilization method for a phosphorylated coenzyme in a dry state,comprising: allowing at least a substance for suppressingdephosphorylation reaction of a phosphorylated coenzyme to coexist witha phosphorylated coenzyme in a solution; and drying the solution;

(3) the stabilization method according to the above-mentioned item (1)or (2), wherein the substance for suppressing dephosphorylation reactionis a sugar and/or a sugar alcohol;

(4) the stabilization method according to the above-mentioned item (1)or (2), wherein the substance for suppressing dephosphorylation reactionis a polysaccharide containing glucose as a constituent;

(5) the stabilization method according to the above-mentioned item (4),wherein the substance for suppressing dephosphorylation reaction is adisaccharide containing glucose as a constituent;

(6) the stabilization method according to the above-mentioned item (5),wherein the substance for suppressing dephosphorylation reaction is oneor a combination of two or more selected from the group consisting oflactose, trehalose, maltose, and sucrose;

(7) the stabilization method according to the above-mentioned item (2),wherein the drying is performed by air drying;

(8) the stabilization method according to any one of the above-mentioneditems (1) to (7), wherein the phosphorylated coenzyme is nicotinamideadenine dinucleotide phosphate (NADP);

(9) a stabilized composition of a phosphorylated coenzyme, comprising:at least a substance for suppressing dephosphorylation reaction of aphosphorylated coenzyme and a phosphorylated coenzyme;

(10) a stabilized composition of a phosphorylated coenzyme in a drystate, comprising at least a substance for suppressing dephosphorylationreaction of the phosphorylated coenzyme and a phosphorylated coenzyme;

(11) the stabilized composition according to the above-mentioned item(9) or (10), wherein the substance for suppressing dephosphorylationreaction is a sugar and/or a sugar alcohol;

(12) the stabilized composition according to the above-mentioned item(9) or (10), wherein the substance for suppressing dephosphorylationreaction is a polysaccharide containing glucose as a constituent;

(13) the stabilized composition according to the above-mentioned item(12), wherein the substance for suppressing dephosphorylation reactionis a disaccharide containing glucose as a constituent;

(14) the stabilized composition according to the above-mentioned item(13), wherein the substance for suppressing dephosphorylation reactionis one or a combination of two or more selected from the groupconsisting of lactose, trehalose, maltose, and sucrose;

(15) the stabilized composition according to any one of theabove-mentioned items (9) to (14), wherein the phosphorylated coenzymeis nicotinamide adenine dinucleotide phosphate (NADP);

(16) a use of a substance for suppressing dephosphorylation reaction forstabilizing a phosphorylated coenzyme for a prolonged period;

(17) a use of a polysaccharide containing glucose as a constituent forpreventing at least a phosphorylated coenzyme from dephosphorylationreaction;

(18) a preservation method for a phosphorylated coenzyme for a prolongedperiod, wherein the preservation method utilizes at least a substancefor suppressing dephosphorylation reaction of the phosphorylatedcoenzyme;

(19) a stabilization method for a phosphorylated coenzyme forenzyme-cycling, comprising allowing a phosphorylated coenzyme to coexistwith at least a substance for suppressing dephosphorylation reaction ofthe phosphorylated coenzyme;

(20) a stabilization method for a phosphorylated coenzyme forenzyme-cycling, comprising allowing only a phosphorylated coenzyme tocoexist with at least a substance for suppressing dephosphorylationreaction of the phosphorylated coenzyme;

(21) the stabilization method according to the above-mentioned item (19)or (20), wherein the stabilization method for a phosphorylated coenzymefor enzyme-cycling is a stabilization method in which 70% or more of thephosphorylated coenzyme remains after preserving at 32° C. for threemonths and a ratio of a dephosphorylated coenzyme in the remainingphosphorylated coenzyme is less than 1%;

(22) the stabilization method according to the above-mentioned item (19)or (20), wherein the stabilization method for a phosphorylated coenzymefor enzyme-cycling is a stabilization method in which 70% or more of thephosphorylated coenzyme remains after preserving at 32° C. for sixmonths and a ratio of a dephosphorylated coenzyme in the remainingphosphorylated coenzyme is less than 1%;

(23) the stabilization method according to the above-mentioned item (19)or (20), wherein the stabilization method for a phosphorylated coenzymefor enzyme-cycling is a stabilization method in which 70% or more of thephosphorylated coenzyme remains after preserving at 32° C. for one yearand a ratio of a dephosphorylated coenzyme in the remainingphosphorylated coenzyme is less than 1%;

(24) the stabilization method according to any one of theabove-mentioned items (19) to (23), wherein the phosphorylated coenzymeincludes nicotinamide adenine dinucleotide phosphates;

(25) the stabilization method according to the above-mentioned item(24), wherein the nicotinamide adenine dinucleotide phosphates arenicotinamide adenine dinucleotide phosphate and/or thionicotinamideadenine dinucleotide phosphate;

(26) the stabilization method according to the above-mentioned item(25), wherein the nicotinamide adenine dinucleotide phosphate and/orthionicotinamide adenine dinucleotide phosphate include/includesnicotinamide adenine dinucleotide phosphate;

(27) the stabilization method according to the above-mentioned item(25), wherein the nicotinamide adenine dinucleotide phosphate and/orthionicotinamide adenine dinucleotide phosphate include/includesthionicotinamide adenine dinucleotide phosphate;

(28) the stabilization method according to any one of theabove-mentioned items (19) to (27), wherein at least the substance forsuppressing dephosphorylation reaction of the phosphorylated coenzyme isa polysaccharide that contains glucose as a constituent;

(29) the stabilization method according to the above-mentioned item(28), wherein the polysaccharide that contains glucose as a constituentis a disaccharide that contains glucose as a constituent;

(30) the stabilization method according to the above-mentioned item(29), wherein the disaccharide that contains glucose as a constituent isone or a combination of two or more selected from the group consistingof maltose, sucrose, lactose, and trehalose;

(31) the stabilization method according to the above-mentioned item(30), wherein the disaccharide that contains glucose as a constituent isone or a combination of two or more selected from the group consistingof maltose, trehalose, and lactose;

(32) the stabilization method according to the above-mentioned item(30), wherein the disaccharide that contains glucose as a constituent ismaltose and/or lactose;

(33) the stabilization method according to the above-mentioned item(30), wherein the disaccharide that contains glucose as a constituent islactose and/or trehalose;

(34) the stabilization method according to the above-mentioned item(30), wherein the disaccharide that contains glucose as a constituent ismaltose and/or trehalose;

(35) the stabilization method according to the above-mentioned item(30), wherein the disaccharide that contains glucose as a constituent islactose and/or sucrose;

(36) the stabilization method according to the above-mentioned item(30), wherein the disaccharide that contains glucose as a constituent ismaltose and/or sucrose;

(37) the stabilization method according to the above-mentioned item(30), wherein the disaccharide that contains glucose as a constituent islactose;

(38) the stabilization method according to the above-mentioned item(30), wherein the disaccharide that contains glucose as a constituent ismaltose;

(39) the stabilization method according to the above-mentioned item(30), wherein the disaccharide that contains glucose as a constituent issucrose;

(40) the stabilization method according to the above-mentioned item(30), wherein the disaccharide that contains glucose as a constituent istrehalose;

(41) the method of measuring the activity of alkaline phosphatase,wherein the stabilized composition according to any one of theabove-mentioned items (9) to (15) is used;

(42) a flow-through simplified immunoassay device, comprising asolution-sending device which allows addition of an enzyme substrate tobe automatically performed after allowing an enzyme-labeled reagent topermeate through an antibody- or antigen-immobilized membrane;

(43) the simplified immunoassay device according to the above-mentioneditem (42), wherein the solution-sending device uses a water-solublefilter;

(44) the simplified immunoassay device according to the above-mentioneditem (42) or (43), wherein the solution-sending device uses a siphon;

(45) the simplified immunoassay device according to any one of theabove-mentioned items (42) to (44), wherein the enzyme substrate is asolution containing the stabilized composition according to any one ofthe above-mentioned items (9) to (15);

(46) the simplified immunoassay device according to any one of theabove-mentioned items (42) to (45), wherein the simplified immunoassaydevice prevents a washing solution from being added after allowing theenzyme-labeled reagent to permeate through the antibody- orantigen-immobilized membrane and before automatically adding the enzymesubstrate;

(47) an enzymatic immunoassay using the method of measuring the activityof alkaline phosphatase according to the above-mentioned item (41),wherein an antibody against a microbial ribosomal protein is used;

(48) the enzymatic immunoassay according to the above-mentioned item(47), wherein the microorganism is one or more selected from Mycoplasmapneumoniae, Hemophilus influenza, Streptococcus pneumoniae, Chlamydiapneumoniae, Chlamydia trachomatis, and Legionella pneumophila;

(49) the enzymatic immunoassay according to the above-mentioned item(47) or (48), wherein the ribosomal protein is 1.7/L12;

(50) the simplified immunoassay device according to any one of theabove-mentioned items (42) to (46), wherein an antibody against theribosomal protein of the microorganism is used;

(51) the simplified immunoassay device according to the above-mentioneditem (50) using the enzymatic immunoassay according to theabove-mentioned item (48), wherein the microorganism is one or moreselected from the group consisting of Mycoplasma pneumoniae, Hemophilusinfluenza, Streptococcus pneumoniae, Chlamydia pneumoniae, Chlamydiatrachomatis, and Legionella pneumophila is used;

(52) the enzymatic immunoassay according to the above-mentioned item(50) or (51), using the enzymatic immunoassay according to theabove-mentioned item (49), wherein the ribosomal protein is L7/L12;

(53) the stabilization method according to any one of theabove-mentioned items (1) to (8), further comprising allowing a citrateto coexist;

(54) the stabilization method according to the above-mentioned item(53), wherein the citrate is sodium citrate;

(55) the stabilized composition according to any one of theabove-mentioned items (9) to (15), further comprising allowing a citrateto coexist;

(56) the stabilized composition according to the above-mentioned item(55), wherein the citrate is sodium citrate;

(57) the use according to the above-mentioned item (16) or (17), whereina citrate is used;

(58) the use according to the above-mentioned item (57), wherein thecitrate is sodium citrate;

(59) the preservation method for phosphorylated coenzyme for a prolongedperiod according to the above-mentioned item (18), wherein a citrate isused;

(60) the stabilization method according to any one of theabove-mentioned items (19) to (40), wherein a citrate is further used;

(61) the stabilization method according to the above-mentioned item(60), wherein the citrate is sodium citrate;

(62) a method of measuring the activity of alkaline phosphatase, whereinthe stabilized composition according to any one of the above-mentioneditems (55) to (56) is used;

(63) the simplified immunoassay device as described in any one of theabove-mentioned items (42) to (44), wherein the enzyme substrate is asolution containing the stabilized composition according to theabove-mentioned item (55) or (56);

(64) the simplified immunoassay device according to the above-mentioneditem (63), wherein a washing solution is not added after allowing theenzyme-labeled reagent to permeate through the antibody- orantigen-immobilized membrane and before automatically adding the enzymesubstrate;

(65) an enzymatic immunoassay using the method of measuring the activityof alkaline phosphatase according to the above-mentioned item (62),wherein an antibody against a microbial ribosomal protein is used;

(66) the enzymatic immunoassay according to the above-mentioned item(65), wherein the microorganisms is one or more selected from the groupconsisting of Mycoplasma pneumoniae, Hemophilus influenza, Streptococcuspneumoniae, Chlamydia pneumoniae, Chlamydia trachomatis, and Legionellapneumophila;

(67) the enzymatic immunoassay according to the above-mentioned item(66) or (67), wherein the ribosomal protein is L7/L12;

(68) the simplified immunoassay device according to the above-mentioneditem (63) or (64), wherein an antibody against the ribosomal protein ofthe microorganism is used;

(69) the simplified immunoassay device according to the above-mentioneditem (68), wherein the microorganism is one or more selected from thegroup consisting of Mycoplasma pneumoniae, Hemophilus influenza,Streptococcus pneumoniae, Chlamydia pneumoniae, Chlamydia trachomatis,and Legionella pneumophila is used;

(70) the enzymatic immunoassay according to the above-mentioned item(50) or (51), wherein the ribosomal protein is L7/L12;

(71) the stabilization method according to the above-mentioned item (1),wherein at least a substance for suppressing dephosphorylation reactionof the phosphorylated coenzyme coexists only with a phosphorylatedcoenzyme;

(72) the stabilization method according to the above-mentioned item (2),wherein at least a substance for suppressing dephosphorylation reactionof the phosphorylated coenzyme coexists only with a phosphorylatedcoenzyme;

(73) the stabilization method according to the above-mentioned item (71)or (72), wherein the substance for suppressing dephosphorylationreaction is a sugar and/or a sugar alcohol;

(74) the stabilization method according to the above-mentioned item (71)or (72), wherein the substance for suppressing dephosphorylationreaction is a polysaccharide containing glucose as a constituent;

(75) the stabilization method according to the above-mentioned item(74), wherein the substance for suppressing dephosphorylation reactionis a disaccharide containing glucose as a constituent;

(76) the stabilization method according to the above-mentioned item(75), wherein the substance for suppressing dephosphorylation reactionis one or a combination of two or more selected from the groupconsisting of lactose, trehalose, maltose, and sucrose;

(77) the stabilization method according to the above-mentioned item(72), wherein the drying is performed by air drying;

(78) the stabilization method according to any one of items (71) to(77), wherein the phosphorylated coenzyme is nicotinamide adeninedinucleotide phosphate (NADP);

(79) the stabilized composition according to the above-mentioned item(9), comprising only at least a substance for suppressingdephosphorylation reaction of a phosphorylated coenzyme and aphosphorylated coenzyme;

(80) the stabilized composition according to the above-mentioned item(10), comprising only at least a substance for suppressingdephosphorylation reaction of a phosphorylated coenzyme and aphosphorylated coenzyme;

(81) the stabilized composition according to the above-mentioned item(79) or (80), wherein the substance for suppressing dephosphorylationreaction is a sugar and/or a sugar alcohol;

(82) the stabilized composition according to the above-mentioned item(79) or (80), wherein the substance for suppressing dephosphorylationreaction is a polysaccharide containing glucose as a constituent;

(83) the stabilized composition according to the above-mentioned item(82), wherein the substance for suppressing dephosphorylation reactionis a disaccharide containing glucose as a constituent;

(84) the stabilized composition according to the above-mentioned item(83), wherein the substance for suppressing dephosphorylation reactionis one or a combination of two or more selected from the groupconsisting of lactose, trehalose, maltose, and sucrose;

(85) the stabilized composition according to any one of theabove-mentioned items (79) to (84), wherein the phosphorylated coenzymeis nicotinamide adenine dinucleotide phosphate (NADP);

(86) a method of measuring the activity of alkaline phosphatase, whereinthe stabilized composition according to any one of the above-mentioneditems (79) to (85) is used;

(87) the simplified immunoassay device as described in any one of theabove-mentioned items (42) to (44), wherein the enzyme substrate is asolution containing the stabilized composition according to any one ofthe above-mentioned items (79) to (85);

(88) the stabilization method according to any one of theabove-mentioned items (71) to (78), further including allowing a citrateto coexist;

(89) the stabilization method according to the above-mentioned item(88), wherein the citrate is sodium citrate;

(90) the stabilized composition according to any one of the above items(79) to (85), wherein a citrate further coexist;

(91) the stabilized composition according to the above-mentioned item(90), wherein the citrate is sodium citrate;

(92) a use of a substance for suppressing dephosphorylation reaction forstabilizing a phosphorylated coenzyme for a prolonged period;

(93) a use of only a polysaccharide containing glucose as a constituentand citric acid for preventing at least a phosphorylated coenzyme fromdephosphorylation reaction;

(94) the use according to the above-mentioned item (92) or (93), whereinthe citrate is sodium citrate;

(95) a preservation method for phosphorylated coenzyme for a prolongedperiod, wherein at least a substance for suppressing dephosphorylationreaction of the phosphorylated coenzyme is used; and

(96) a preservation method for phosphorylated coenzyme for a prolongedperiod, wherein only at least a substance for suppressingdephosphorylation reaction of the phosphorylated coenzyme and a citrateare used.

Effects of the Invention

The stabilization method of the present invention exhibits an effect ofstabilizing a coenzyme at room temperature for a prolonged period, forexample, when the stabilization method is used for measuring theactivity of ALP, or the like. Therefore, it is very useful for achievingthe practical use of a test reagent using a coenzyme in a POC testreagent or the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing showing a flow-through-type simplified immunoassaydevice of the present invention, which is provided with asolution-sending device so that an enzyme substrate is automaticallyadded after allowing an enzyme-labeled reagent to permeate through anantibody- or antigen-immobilized membrane.

FIG. 2 is a drawing showing a flow-through simplified immunoassay deviceof the present invention, which is provided with a siphon-typesolution-sending device so that an enzyme substrate is automaticallyadded after allowing an enzyme-labeled reagent to permeate through anantibody- or antigen-immobilized membrane.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

A preferable example of the phosphorylated coenzyme of the presentinvention is one or a combination of two or more coenzymes selected fromthe group consisting of NADP, thio-NADP, acetyl-NADP, deamino-NADP, anddeamide-NADP. NADP or NADPH is more preferable, and NADP is particularlypreferable. The purity of the coenzyme is higher the better. Anycommercial product may be used without modification if it is of highpurity. If necessary, commercial coenzyme powder dissolved in a suitablesolvent and purified by column purification or the like may be used.Impurities, which must be taken into consideration, includeunphosphorylated coenzymes and the like. The amount of theunphosphorylated coenzyme, that is, the impurities, is preferably 1% orless, more preferably 0.5% or less, particularly preferable 0.1% or lesswith respect to the amount of the phosphorylated coenzyme provided as atarget of the stabilization in the present invention.

Substances that inhibit the dephosphorylation reaction in the presentinvention are not particularly limited as far as they have effects toprevent a phosphoric group bound to position 2 of ribose present in atleast the structure of the above phosphorylated coenzyme fromdissociating therefrom. In addition to such effects, the substances mayhave other effects. The other effects include those for preventingdegradation of the binding between a ribose portion and a base portion.Specific examples of the substances include preferably sugars and/orsugar alcohols, particularly preferably sugars.

As the sugars, polysaccharides are preferable. For example,polysaccharides containing glucose in their structures are particularlypreferable. Specific examples of the polysaccharides include sucrose,trehalose, lactose, maltose, α-cyclodextrin, β-cyclodextrin,γ-cyclodextrin, methyl-γ-cyclodextrin, hydroxypropylcyclodextrin,maltodextrin, maltotriose, maltotetraose, maltopentaose, maltohexaose,maltoheptaose, isomaltose, isomaltotriose, isomaltotetraose,isomaltopentaose, isomaltohexaose, panose, isopanose, sophorose,sophorotriose, sophorotetraose, sophoropentaose, sophorohexaose,kojibiose, laminaribiose, laminaritriose, laminaritetraose,laminaripentaose, nigerose, nigerotriose, cellobiose, cellotiose,cellotetraose, cellopentaose, cellohexaose, celloheptaose, gentiobiose,gentiobiose, gentiotetraose, gentiopentaose, gentiohexaose,neotrehalose, isotrehalose, vicianose, isoprimeverose, sambubiose,primeverose, lycotetraose, solabiose, melibiose, manninotriose,verbascotetraose, lycobiose, lycotriose, epiceilobiose, turanose,maltulose, isokestose, erlose, kestose, planteose, planteotetraose,raffinose, stachyose, verbascose, ajugose, lychnose, scillabiose,neohesperidose, rutinose, chacotriose, solatriose, fucosidolactose,difucolactose, strophantobiose, strophantotriose, digyranidobiose,digyranidotriose, odorotriose, trehalosamine, lacto-N-triose II,gentianose, neokestose, melezitose, neobifurcose, nystose, bifurcose,lycoricine, epigentiobiose, isolychnase, umbelliferose, sesamose,lacto-N-tetraose, lacto-N-neotetraose, cellobio uronic acid,N-acetylneuraminolactose I, N-acetylneuraminolactose II,di-N-acetylneuraminolactose, lactopentasaccharide a,lactopentasaccharide b, lactopentasaccharide c, glucosylmannitol,diglucosylmannitol,galactofuranosyl-glucosyl-galactofuranosyl-galactosyl-ribitol, amylose,glycogen, glucan, scierotan, dextran, nigeran, pstulan, pullulan,laminaran, rinaken, inulin, and a polysaccharide antigen on a surface ofa bacterium.

Disaccharides that contain glucose in their structures are furtherpreferable. Optimally, one or a combination of two or more selected fromthe group consisting of lactose, trehalose, maltose, and sucrose ispreferable. In particular, lactose is preferable. Furthermore, inanother aspect, trehalose may be preferable. In still another aspect,maltose may be preferable. In an alternate aspect, sucrose may bepreferable. A preferable combination is of lactose or trehalose with anyof other dissacharides containing glucose in their structures, forexample, it is preferable to add to lactose a saccharide of one or acombination of two or more selected from trehalose, maltose, and sucroseor add to trehalose a saccharide of one or a combination of two or moreselected from lactose, maltose, and sucrose. The ratio for thecombination is not particularly limited as far as a desired effect isexerted. In view of convenience, however, one is defined as 1 and theother is defined as preferably an integral multiplication selected from1 to 100, more preferably an integral multiplication selected from 1 to10, further preferably an integral multiplication selected from 1 to 5,particularly preferably an integral multiplication selected from 1 to 3,extremely preferably 1 or 2 times, most preferably 1 time (=equalamount) in weight, volume, or mole.

The concentration range of the substance that inhibits thedephosphorylation reaction during preservation is not particularlylimited, and the upper limit thereof may be any of concentrations as faras it is equal to or less than a saturated solubility concentrationthereof. In contrast, the lower limit is preferably 0.1% or more, morepreferably 0.5% or more, particularly preferably 1% or more.

The stabilization of the phosphorylated coenzyme in the presentinvention means the state of retaining the high purity of thephosphorylated coenzyme during its preservation period. Specifically, itmeans that the phosphorylated coenzyme is retained at high purity sothat the content thereof is sufficient to be provided as an alkalinephosphatase substrate and an increase in background can be held downduring the preservation period. More specifically, for example, thestate in which the amount of the phosphorylated coenzyme may be 70% ormore and the rate of an increase in unphosphorylated coenzyme to theamount of the phosphorylated coenzyme at the initial stage of thepreservation may be kept at 1% or less for a prolonged period. Morepreferably, the state in which the amount of the phosphorylated coenzymemay be 80% or more and the rate of an increase in unphosphorylatedcoenzyme to the amount of the phosphorylated coenzyme at the initialstage of the preservation may be kept at 1% or less for a prolongedperiod. The preferable prolonged period may be three, six, or twelvemonths or more, and among them, 6 months or more is a particularlypreferable period.

The stabilization method for a phosphorylated coenzyme of the presentinvention has a characteristic feature in that at least a phosphorylatedcoenzyme and a substance for suppressing dephosphorylation reaction areallowed to coexist with each other. The ratio of the phosphorylatedcoenzyme to the substance for suppressing dephosphorylation reaction incoexistence is not particularly limited as far as the phosphorylatedcoenzyme is sufficiently stabilized under the desired preservationconditions and the function of the phosphorylated coenzyme is notinhibited under the desired sample-measurement conditions. For instance,a preferable ratio is from 10⁷ molecules to 0.001 molecules, preferablyfrom 10⁶ molecules to 0.001 molecules, optimally 10⁶ molecules to 0.002molecules of the substance for suppressing dephosphorylation reaction toone molecule of the phosphorylated coenzyme.

Furthermore, a stabilization method using an appropriate buffer as anadditive can be mentioned. The buffer may be added with an appropriateconcentration of the phosphorylated coenzyme. The concentration range ofthe phosphorylated coenzyme is not particularly limited, but the lowerlimit thereof is preferably 0.1 mM or more, more preferably 0.5 mM ormore, particularly preferably 1 mM or more. In addition, the upper limitof the phosphorylated Coenzyme is preferably 1 M or less, morepreferably 500 mM or less, particularly preferably 200 mM or less. Thebuffer is not particularly limited, so it may be any of buffers.However, the buffer may be preferably one or a combination of two ormore selected from the group consisting of: an oxalic acid buffer, anethylenediamine tetraacetic acid buffer, a maleic acid buffer, anaspartic acid buffer, a phosphate buffer, an asparagine buffer, aglycine buffer, a pyruvic acid buffer, a pyrophosphate buffer, a malonicacid buffer, a phthalate buffer, a fumaric acid buffer, a tartaric acidbuffer, a citrate buffer, a furancarboxylic acid buffer, a β-alaninebuffer, a β: β′-dimethyl glutaric acid buffer, a formic acid buffer, alactic acid buffer, a γ-aminobutyric acid buffer, a barbituric acidbuffer, a benzoic acid buffer, a succinic acid buffer, a E-aminocaproicacid buffer, an acetic acid buffer, a propionic acid buffer, a malicacid buffer, a pyridine buffer, a histidine buffer, a cacodylic acidbuffer, a carbonic acid buffer, a hydroxyimidazole buffer, a glycerolphosphate buffer, an ethylenediamine buffer, an imidazole buffer, anarsenic acid buffer, a 2,4,6-collidine buffer, a 1-, 2-, or 4-methylimidazole buffer, an N-ethyl morpholine buffer, a veronal buffer, abarbital buffer, a 2,4-dimethyl imidazole buffer, a morpholine buffer,an N-ethyl morpholine buffer, a 2-amino-2-methyl-1,3-propanediol buffer,a 2-amino-2-ethyl-1,3-propanediol buffer, a diethanolamine buffer, a4-aminopyridine buffer, a serine buffer, a boric acid buffer, an ammoniabuffer, an ethanolamine buffer, an ephedrine buffer, a hydroxyprolinebuffer, a 2-amino-2-methyl-1-propanol buffer, a leucine buffer, atrimethyl buffer, an α-alanine buffer, a n-propyl alcohol buffer, amethylamine buffer, an ethylamine buffer, a n-butylamine buffer, atriethylamine buffer, a dimethylamine buffer, a hexamethylenediaminebuffer, a piperidine buffer, a p-toluenesulfonic acid buffer, a Trisbuffer, a glycylglycine buffer, and a GTA buffer; and a Good buffer suchas an MES buffer, a Bis-Tris buffer, an ADA buffer, a PIPES buffer, anACES buffer, an MOPSO buffer, a BES buffer, an MOPS buffer, a TESbuffer, an HEPES buffer, a DIPSO buffer, a TAPSO buffer, a POPSO buffer,an HEPPSO buffer, an EPPS buffer, a Tricine buffer, a Bicine buffer, aTAPS buffer, a CHES buffer, a CAPSO buffer, and a CAPS buffer, andpreferably selected from the group consisting of a citrate buffer, adiethanolamine buffer, a Tris buffer, a glycine buffer, an acetic acidbuffer, a carbonic acid buffer, an imidazole buffer, a 1-, 2-, or4-methyl imidazole buffer, a veronal buffer, a barbital buffer, a2,4-dimethyl imidazole buffer, a 2-amino-2-ethyl-1,3-propanediol buffer,a boric acid buffer, a triethylamine buffer, a dimethylamine buffer, anda Good buffer. Optimally, a citrate buffer or a diethanolamine buffer ispreferable. The pH of the buffer is preferably from neutral to alkalinewhen the phosphorylated coenzyme is a reduced type, preferably from pH 7to 12, optimally pH 7.5 to 11. In addition, when the phosphorylatedcoenzyme is an acidic type, it is preferably from neutral to acidic,preferably pH 2 to 7.5, optimally pH 3 to 7.

For the stabilization method of the present invention, a preferablemethod may be one in which a preservative such as sodium azide orproclin is added for improving a general preservation property. A methodin which a dried preservation composition is obtained by drying any ofthose solutions may be also mentioned.

The stabilized composition of the present invention may be one preparedas a composition containing at least a substance for suppressingdephosphorylation reaction of a phosphorylated coenzyme and aphosphorylated coenzyme in accordance with the stabilization methoddescribed above. A composition prepared by allowing the composition tofurther include the above buffer, the above preservative, or the like isalso preferable. The composition may be of a liquid or dried form,preferably of a dried form. The dried composition may be prepared bymixing dried products or preferably prepared by preparing a mixture insolution and then drying the mixture. Examples of the drying methodinclude, but not particularly limited to, lyophilization, air-drying,ustulation, and vacuum drying, preferably lyophilization and air-drying,particularly preferably air-drying. The temperature at drying is notparticularly limited as far as it is under conditions that do notdecompose a phosphorylated coenzyme. It may be −80° C. to 100° C.,preferably −50° C. to 80° C., optimally −50° C. to 70° C. Furthermore,the drying time may be suitably defined using the dryness of moisture asan index depending on the drying conditions. The index of the moisturedryness is more preferable as the content of moisture is lower,preferably 10% or less, more preferably 5% or less, optimally 3% orless, extremely preferably 1% or less.

The preservation temperature of the stabilized composition of thepresent invention is not particularly limited as far as it is defineddepending on the purpose. However, the upper limit of the temperature ispreferably 50° C. or less, more preferably 40° C. or less, still morepreferably 37° C. or less, particularly preferably 32° C. or less, mostpreferably 30° C. or less. There is no need of specifically defining thelower limit of the temperature. From a practical viewpoint, it ispreferably −20° C. or more, more preferably −10° C. or more, still morepreferably 0° C. or more, particularly preferably 4° C. or more, mostpreferably 10° C. or more. The characteristic feature of thestabilization method of the present invention is that a stablepreservation can be attained at temperatures ranging from −20° C. to 40°C., from 4° C. to 37° C., or from 10° C. and 32° C.

In this way, by the use of a coenzyme that can be preserved at normaltemperature for a prolonged period, it becomes possible to provide ahigh-sensitive assay for ALP activity, which can be also retained atroom temperature for a prolonged period, for the first time. In theassay for ALP activity, for example, NADP being preserved in dried stateis re-dissolved in a reaction solution containing 12α hydroxysteroiddehydrogenase, diaphorase, cholic acid, nitroblue-tetrazolium dissolvedin a buffer and ALP to be measured is then added to the reactionsolution, followed by incubating at room temperature or 37° C. for apredetermined period. The amount of pigments accumulated in the solutionmay be determined as an increment per unit time or an accumulatedabsolute amount by a spectrophotometer or visual observation. For theusage of such an assay for ALP activity, as described above, it can becommonly used for a detection reagent, a detection kit containing such adetection reagent, and so on in any fields of examination. An examplethereof includes a reagent for detecting E. coli O-157, which can bepreserved at room temperature. Furthermore, the kit may be one composedof a plate on which an O-157 gene probe is immobilized, a sampleextract, a digoxigenin-labeled nucleic acid, an anti-digoxigeninALP-labeled antibody, a washing solution, an NADP-dried product for ALPdetection, an enzyme-dried product for ALP detection, and an ALPdetection reagent-dissolving solution, which can be preserved as areagent at room temperature.

For describing the method simply, an O-157 gene-immobilized plate isprepared by immobilizing a probe nucleic acid for detecting an E. coliO-157 gene on a plastic plate. A sample extract obtained from thenucleic acid of E. coli O-157 as a sample is dispensed into the plate,and then incubated at room temperature or 37° C. for a predeterminedperiod. The plate is washed with a washing solution and then added witha digoxigenin-labeled nucleic acid, which has a sequence complement tothat of the E. coli O-157 gene and has a region different from that ofthe probe on the plate solid phase, followed by incubation at roomtemperature or 37° C. for a predetermined period. The plate is washedand then added with an anti-digoxigenin ALP-labeled antibody, followedby incubation at room temperature or 37° C. for a predetermined period.After washing to the plate, for detecting an anti-digoxigeninALP-labeled antibody-digoxigenin-nucleic acid complex formed on thesolid phase, an enzyme-dried product for ALP detection containing 12αhydroxysteroid dehydrogenase, diaphorase, cholic acid, and nitrobluetetrazolium and a reaction solution for ALP detection prepared byre-dissolving an NADP-dried product for ALP detection in an ALPdetection reagent-dissolving solution were added to the plate in certainamounts, followed by incubation at room temperature or 37° C. for apredetermined period. The presence of E. coli O-157 in the sample isconfirmed by reading out using an optical device such as a plate readeror visually determining the amount of a color-developed formazan pigmenton the plate. If E. coli O-157 is present, the ALP activity supported bythe O-157 gene shows a strong color development. In contrast, if E. coliO-157 is absent, the color development does not occur or is extremelysmall.

Furthermore, the use of a substance for suppressing dephosphorylationreaction for stabilizing the phosphorylated enzyme for a prolongedperiod, the use of polysaccharides containing glucose as a constituentfor preventing the dephosphorylation reaction of at least aphosphorylated coenzyme, and a method of preserving a phosphorylatedcoenzyme for a prolonged period using at least a substance forsuppressing dephosphorylation reaction of the phosphorylated coenzymeare also within the scope of the present invention.

The coenzyme of the present invention stabilized as described above canbe applied to a simplified immunoassay device using an enzyme cyclingmethod, so the simplified immunoassay device can be also within thescope of the present invention. The principle of the simplifiedimmunoassay device is preferably an immunochromatographic method or aflow-through method with the flow-through method being preferable. Thesimplified immunoassay device may be of, for example, in the case of theflow-through method, immobilizing an antibody or the like having abinding ability to a substance to be provided as a measurement object inadvance on a membrane; mounting such a membrane on a material havingwater-absorption ability; and, for allowing the membrane to capture themeasurement object, carrying out an antigen-antibody reaction bydropping onto the membrane a liquid in which the measurement object ispreviously reacted with another enzyme-labeled antibody having thebinding ability to the measurement object, or dropping a liquidcontaining the measurement object and dropping an enzyme-labeledantibody solution after the liquid is substantially depleted from themembrane by absorption of the liquid. Subsequently, after the liquid issubstantially depleted from the membrane, a washing solution is droppedas a washing operation, and similarly after the washing solution isdepleted from the membrane, an enzyme substrate solution is added. Ingeneral, the enzyme in the complex formed on the membrane is reactedwith the enzyme substrate to confirm the formation of a pigment on themembrane.

In this case, for efficiently forming a complex containing theenzyme-labeled antibody on the membrane, the duration of contacting theenzyme-labeled antibody solution with the membrane is longer the better.In contrast, in view of operability, the washing solution and the enzymesubstrate solution may preferably permeate through the membrane within ashorter period. Therefore, it is a common method in which the substanceprovided as a measurement object and the enzyme-labeled antibody may beadded with a predetermined time interval, and the washing solution, theenzyme substrate solution, and so on are added with a shorter intervalin that order. This method has a problem in the viewpoint ofoperability.

In the present invention, as a method of improving a plurality ofoperations with such time intervals, a plurality of liquids is added toa simplified immunoassay device substantially at the same time and theliquids are then added to a membrane in order by an automaticsolution-sending device incorporated in the inside of the device. Inparticular, when an enzyme-labeled antibody and an enzyme substratesolution are mixed on the membrane, a color development may occurwithout depending on the presence or absence of a substance provided asa measurement object, thereby causing a nonspecific background.Therefore, for avoiding such a trouble, the inventors of the presentinvention have invented a simplified immunoassay device in which anautomatic solution-sending device for preventing them from contactingwith each other, as much as possible. The automatic solution-sendingdevice provides a time lag so that, when both an enzyme-labeled antibodysolution and an enzyme substrate solution is charged into the devicesimultaneously, first the enzyme-labeled antibody solution permeatesthrough the membrane for, a predetermined period, and the enzymesubstrate solution is then added after the permeation through themembrane is substantially completed. When the time lag is provided,anything can be used. For example, there is a method in which, when theenzyme substrate solution is added, the solution may be blocked by anwater-soluble filter before the liquid approaches to the membrane andthen the enzyme substrate solution is added to the membrane after apredetermined time period during which the water-soluble filter can bedissolved.

The water-soluble filter allows an enzyme substrate solution not to flowout for a predetermined period and allows it to flow out after thewater-soluble filter is dissolved by the enzyme substrate solution at acertain point of time. Therefore, any filter can be used as far as itdoes not substantially influence an enzyme reaction. For example, afilm-shaped filter having water-soluble property is preferable.

Examples of the film-shaped filter having water-soluble property includea polyvinyl alcohol membrane, a pullulan membrane, and a hydroxypropylcellulose membrane. Among them, preferable is the polyvinyl alcoholmembrane. Any of these water-soluble filters is mounted on the lowerpart of a cylindrical enzyme substrate solution adding vessel. When theenzyme substrate solution is added, the solution does not flow out for apredetermined time while dissolving the water-soluble filter, and afterthat, the solution begins to flow. The time period that does not allowthe solution to flow is in the range of 10 seconds to 10 minutes,preferably 30 seconds to 5 minutes.

Furthermore, for the automatic solution-sending device of the presentinvention, a method using a siphon may be employed. The siphon of thepresent invention is a tube having an opening at the lower part of anenzyme substrate solution adding vessel, where the tube has a structurethat extends from the opening to the upper part and then extends to thelower part, while the other opening of the tube is located at a portioncommunicating with an observation window. Besides, the siphon may beprovided with a filter for controlling the flow rate of the enzymesubstrate solution, which is located between the lower part and theupper end of the enzyme substrate solution adding vessel. When an enzymesubstrate solution is added to the enzyme substrate solution addingvessel having a siphon structure, the solution is gradually accumulatedin the lower part of the enzyme substrate solution adding vessel by aflow-rate control filter. Subsequently, the tube is filled with thesolution so as to correspond to the height of the liquid in accordancewith an increase in preservation. When the surface of the preservedsolution becomes higher than the highest position of the tube, thepreserved solution passes through the tube and flows into an observationwindow at once. Therefore, when the enzyme substrate solution is addedto the enzyme substrate solution adding vessel, the solution does notflow for a given period of time and then flows out at once. Any offlow-rate control filters can be used as far as an enzyme substratesolution is allowed to moderately flow out to the lower part of anenzyme substrate solution adding vessel after the enzyme substratesolution is added to the enzyme substrate solution adding vessel and afilter paper on which a water-soluble substance is dried is preferable.The material of such filter paper may be any substance as far as itallows the permeation of a solution after re-dissolving thewater-soluble substance. For examples, the materials include paper,glass, polyester, polystyrene, and nylon. Among them, paper or glass ispreferable. The water-soluble substances include sugars, proteins, andsynthetic polymers. The sugars include macromolecular polysaccharidessuch as dextran, pullulan, and agar; dissacharides such as sucrose,lactose, and maltose; and monosaccharides such as glucose and galactose.Among them, the macromolecular polysaccharides are preferable anddextran is more preferable. The proteins include albumin and globulin.The synthetic polymers include polyethylene glycol,polyvinylpyrrolidone, polyvinyl alcohol, and hydroxypropylcellulose. Theflow rate of the solution may be suitably adjusted by the concentrationof the water-soluble substance in the dry state, and the time periodthat does not allow the solution to flow is 10 seconds to 10 minutes,preferably 30 seconds to 5 minutes.

By using those, it is also easy to add a washing solution by a similarway before the addition of the enzyme substrate solution to theobservation window. However, such an addition is not always required.The addition may be set depending on the background which can berecognized in the absence of a substance to be measured and the requiredsensitivity. For operability and cost effectiveness, it is preferablenot to use the washing solution.

For more detailed description, for example, an influenza antigensuspension is obtained by collecting a liquid wiped out from a nasalcavity of a patient infected with influenza by using a swab or the like,and suspending an influenza antigen attached to the swab in a solutionof an alkaline phosphatase-labeled anti-influenza antibody, whichcontains a surfactant, which serves as an influenza antigen extract, anappropriate buffer, salts, and so on.

An appropriate amount of the influenza antigen suspension is dropped tothe observation window of the simplified immunoassay device and anenzyme-cycling reaction solution is immediately added to the enzymesubstrate solution adding vessel. After several minutes, the presence orabsence of antigen may be visually confirmed by observation of pigmentprecipitation through the observation window, so the presence or absenceof antigen can be determined without any specific operation after theaddition of the influenza antigen suspension and the enzyme-cyclingreaction solution.

Furthermore, for detecting other microorganisms other than influenza, itis also possible to provide a simplified immunoassay having excellentspecificity and detection sensitivity by making a combination of anantibody against a microbial ribosomal protein and an enzyme cyclingmethod as described in WO 00/06603. The ribosomal protein used may beL7/L12. In particular, the simplified immunoassay is effective toMycoplasma pneumoniae, Hemophilus influenza, Streptococcus pneumoniae,Chlamydia pneumoniae, Chlamydia trachomatis, and Legionella pneumophila,on which excellent specificity and binding ability are demanded.Mycoplasma pneumoniae or Legionella pneumophila is preferable, andpreferably it is desirable to use an antibody against Mycoplasmapneumoniae.

EXAMPLES

Hereinafter, the present invention is further described with referenceto examples and comparative examples. However, the present invention isnot restricted by these examples.

Example 1 (1) Preservation of Coenzyme

In a 20 mM sodium citrate buffer (pH 6.5) containing 5% lactose, NADP(manufactured by Oriental Yeast Co., Ltd.) as a coenzyme was dissolvedto be 1.25 mM to prepare a solution. In addition, as a control, asolution was prepared by dissolving NADP in a 20 mM sodium citratebuffer (pH 6.5) so as to be 1.25 mM, and five different solutions intotal were prepared. The solutions were each dispensed into microtubesat an amount of 20 per microtube and dried for 5 hours under theconditions at 30° C. with 15% humidity. After drying, the microtubeswere preserved at 4° C., 25° C., 32° C., 37° C., and 42° C.,respectively, in an aluminum bag containing a desiccant.

(2) Measurement of Background

As an enzyme-cycling reaction solution, a solution containing 100 mMdiethanolamine-HCl (pH 9.5), 0.0125% nitroblue tetrazolium, 1 mM cholicacid, 50 mM sodium chloride, 1 mM magnesium chloride, and 6.6 U/mldiaphorase from Bacillus megaterium (manufactured by Asahikasei PharmaCorporation) was prepared. One milliliter of the solution was added to amicrotube in which NADP bad been preserved in the dry state and the NADPwas then stirred and redissolved therein. In a tube, 500 μl of thecycling reaction solution containing the redissolved NADP was heated at37° C. for 3 minutes and then added with 5 μl of 1777.6 U/ml 12αhydroxysteroid dehydrogenase (manufactured by Asahikasei PharmaCorporation) from Bacillus sphaericus dissolved in 10 mM PIPES (pH 8.5),followed by reacting at 37° C. for 5 minutes. The reaction wasterminated by the addition of 250 μl of a 0.5% sodium dodecylsulfatesolution and the reaction solution was then subjected to the measurementof absorbance at 550 nm.

(3) Measurement of Alkaline Phosphatase Activity

In a tube, 500 μl of the cycling reaction solution containing theredissolved NADP obtained in the above-mentioned item (2) was heated at37° C. for 3 minutes, then simultaneously added with 5 μl of 1777.6 U/ml12α hydroxysteroid dehydrogenase (manufactured by Asahikasei PharmaCorporation) from Bacillus sphaericus dissolved in 10 mM PIPES (pH 8.5)and 5 μl of 50 mU/ml of alkaline phosphatase from calf small intestine(manufactured by Roche Co., Ltd.) and then reacted at 37° C. for 5minutes. The reaction was terminated by the addition of 250 μl of a 0.5%sodium dodecylsulfate solution and the reaction solution was thensubjected to the measurement of absorbance at 550 nm. The values ofbackground obtained in the above-mentioned item (2) under the respectiveexperimental conditions were subtracted from the resulting measurementvalues and the obtained values were then provided as activity valuesobtained by the alkaline phosphatase reaction, respectively.

(4) Measurement of NADP Amount

NADP preserved in the dry state was re-dissolved by the addition of 1 mlof a 10-mM PIPES (pH 8) and a part thereof was then diluted 10-fold intodistilled water. In 1 ml of a solution which was prepared such that theenzyme-cycling reaction solution prepared in the above-mentioned item(2) contained 8 U/ml alkaline phosphatase from calf small intestine, wasadded with 20 μl of a previously-prepared NADP-redissolved solution,followed by reacting at 37° C. for 5 minutes. The reaction wasterminated by the addition of 250 μl of a 0.5% sodium dodecylsulfatesolution and the reaction solution was then subjected to the measurementof absorbance at 550 nm. The measurement value of NADP at the start ofpreservation was defined as 100% and the remaining % was thencalculated.

As a result, as shown in Table 1-1 and Table 1-2, the addition oflactose attained a decrease in background after 3-month preservation ascompared with the experimental control and the ALP activity was alsomeasured. In addition, as shown in Table 1-3, the amount of NADPremained was also retained as compared with the experimental control.

TABLE 1-1 Background Immediately after the 3-month preservation Abs: 550nm start of preservation 4° C. 25° C. 32° C. 37° C. 42° C. 5% lactose0.067 0.071 0.081 0.122 0.224 0.259 Experimental control 0.067 0.1830.386 0.608 1.233 1.956

TABLE 1-2 Measurement value of Immediately after the 3-monthpreservation ALP activity Abs: 550 nm start of preservation 4° C. 25° C.32° C. 37° C. 42° C. 5% lactose 0.276 0.274 0.288 0.262 0.239 0.200Experimental control 0-278 0.287 0.283 0.305 0.210 0.129

TABLE 1-3 Remaining % of Immediately after the 3-month preservation NADPamount start of preservation 4° C. 25° C. 32° C. 37° C. 42° C. 5%lactose 100 99 100 100 91 63 Experimental control 100 91 88 76 43 31

Example 2

An experiment was conducted in a manner similar to Example 1 except thatthree different solutions were prepared by dissolving NADP (manufacturedby Oriental Yeast Co., Ltd.) as a coenzyme so as to be 1.25 mM in 20-mMsodium citrate buffers (pH 6.5) that contained, instead of 5% lactose,10% trehalose, 10% maltose, and 10% sucrose, respectively.

As a result, as shown in Table 2-1 to Table 2-3, each of the conditionsof 10% trehalose, 10% maltose, and 10% sucrose showed good stability ascompared with the experimental control.

TABLE 2-1 Background Immediately after the 3-month preservation Abs: 550nm start of preservation 4° C. 25° C. 32° C. 37° C. 42° C. 10% trehalose0.067 0.066 0.101 0.139 0.588 0.772 10% maltose 0.094 0.093 0.109 0.1460.225 0.292 10% sucrose 0.067 0.068 0.108 0.164 0.622 0.783 Experimentalcontrol 0.067 0.183 0.386 0.608 1.233 1.956

TABLE 2-2 Measurement value of Immediately after the 3-monthpreservation ALP activity Abs: 550 nm start of preservation 4° C. 25° C.32° C. 37° C. 42° C. 10% trehalose 0.276 0.261 0.271 0.263 0.195 0.12810% maltose 0.299 0.302 0.287 0.278 0.240 0.199 10% sucrose 0.276 0.2910.275 0.269 0.188 0.138 Experimental control 0.278 0.287 0.283 0.3050.210 0.129

TABLE 2-3 Remaining % of Immediately after the 3-month preservation NADPamount start of preservation 4° C. 25° C. 32° C. 37° C. 42° C. 10%trehalose 100 96 105 97 75 60 10% maltose 100 97 102 97 74 68 10%sucrose 100 97 89 72 67 46 Experimental control 100 91 88 76 43 31

Example 3

An experiment was conducted in a manner similar to Example 1 except thatsix different solutions were prepared by dissolving NADP (manufacturedby Oriental Yeast Co., Ltd.) as a coenzyme so as to be 1.25 mM in 20-mMsodium citrate buffers (pH 6.5) that contained, instead of 5% lactose,trehalose adjusted to concentrations of 0.1%, 1%, 5%, 10%, 30%, and 60%,respectively.

As a result, as shown in Table 3-1 to Table 3-3, in the range of 0.1% to60% trehalose concentrations, good stability was obtained as comparedwith the experimental control.

TABLE 3-1 Background Immediately after the 3-month preservation Abs: 550nm start of preservation 4° C. 25° C. 32° C. 37° C. 42° C. 0.1%trehalose 0.068 0.130 0.202 0.323 0.888 1.237 1% trehalose 0.067 0.0890.124 0.191 0.765 0.980 5% trehalose 0.066 0.071 0.109 0.160 0.633 0.80010% trehalose 0.067 0.066 0.101 0.139 0.588 0.772 30% trehalose 0.0650.058 0.098 0.152 0.497 0.702 60% trehalose 0.064 0.055 0.111 0.1430.488 0.695 Experimental control 0.067 0.183 0.386 0.608 1.233 1.956

TABLE 3-2 Measurement value of Immediately after the 3-monthpreservation ALP activity Abs: 550 nm start of preservation 4° C. 25° C.32° C. 37° C. 42° C. 0.1% trehalose 0.275 0.276 0.288 0.299 0.200 0.1211% trehalose 0.266 0.262 0.275 0.271 0.188 0.132 5% trehalose 0.2650.270 0.272 0.261 0.202 0.123 10% trehalose 0.276 0.261 0.271 0.2630.195 0.128 30% trehalose 0.280 0.277 0.289 0.274 0.223 0.188 60%trehalose 0.271 0.269 0.276 0.280 0.230 0.174 Experimental control 0.2780.287 0.283 0.305 0.210 0.129

TABLE 3-3 Remaining % of Immediately after the 3-month preservation NADPamount start of preservation 4° C. 25° C. 32° C. 37° C. 42° C. 0.1%trehalose 100 102 102 83 66 40 1% trehalose 100 100 103 89 69 58 5%trehalose 100 99 95 101 69 57 10% trehalose 100 96 105 97 75 60 30%trehalose 100 101 100 98 81 64 60% trehalose 100 96 99 98 79 66Experimental control 100 91 88 76 43 31

Example 4

An experiment was conducted in a manner similar to Example 1 except thatfive different solutions were prepared by dissolving NADP (manufacturedby Oriental Yeast Co., Ltd.) as a coenzyme so as to be 1.25 mM in 20-mMsodium citrate buffers (pH 6.5) that contained, instead of 5% lactose,lactose adjusted to concentrations of 0.1%, 0.5%, 1%, 5%, and 15%,respectively.

As a result, as shown in Table 4-1 to Table 4-3, in the range of 0.1% to15% lactose concentrations, good stability was obtained as compared withthe experimental control.

TABLE 4-1 Background Immediately after the 3-month preservation Abs: 550nm start of preservation 4° C. 25° C. 32° C. 37° C. 42° C. 0.1% lactose0.069 0.090 0.142 0.233 0.488 0.605 0.5% lactose 0.066 0.083 0.112 0.1530.312 0.386 1% lactose 0.065 0.070 0.071 0.131 0.253 0.297 5% lactose0.067 0.071 0.081 0.122 0.224 0.259 15% lactose 0.071 0.065 0.066 0.1110.196 0.240 Experimental control 0.067 0.183 0.386 0.608 1.233 1.956

TABLE 4-2 Measurement value of Immediately after the 3-monthpreservation ALP activity Abs: 550 nm start of preservation 4° C. 25° C.32° C. 37° C. 42° C. 0.1% lactose 0.278 0.278 0.283 0.267 0.206 0.1300.5% lactose 0.295 0.292 0.276 0.231 0.237 0.143 1% lactose 0.276 0.2660.281 0.253 0.254 0.188 5% lactose 0.276 0.274 0.288 0.262 0.239 0.20015% lactose 0.280 0.291 0.290 0.263 0.241 0.213 Experimental control0.278 0.287 0.283 0.305 0.210 0.129

TABLE 4-3 Remaining % of Immediately after the 3-month preservation NADPamount start of preservation 4° C. 25° C. 32° C. 37° C. 42° C. 0.1%lactose 100 95 95 95 77 33 0.5% lactose 100 96 95 93 88 45 1% lactose100 98 103 99 92 58 5% lactose 100 99 100 100 91 63 15% lactose 100 101100 96 97 71 Experimental control 100 91 88 76 43 31

Example 5

An experiment was conducted in a manner similar to Example 1 except thatfour different solutions were prepared by dissolving NADP (manufacturedby Oriental Yeast Co., Ltd.) as a coenzyme so as to be 1.25 mM in 20-mMsodium citrate buffers (pH 6.5) that contained, instead of 5% lactose:condition 1 with 10% lactose and 20% trehalose; condition 2 with 10%lactose and 20% maltose; condition 3 with 20% trehalose and 20% maltose;and condition 4 with 10% lactose, 20% trehalose, and 20% maltose,respectively.

As a result, as shown in Table 5-1 to Table 5-3, each of the conditions1 to 4 showed good stability as compared with the experimental control.

TABLE 5-1 Background Immediately after the 3-month preservation Abs: 550nm start of preservation 4° C. 25° C. 32° C. 37° C. 42° C. Condition 10.065 0.070 0.088 0.124 0.244 0.287 Condition 2 0.071 0.081 0.080 0.1130.192 0.229 Condition 3 0.084 0.079 0.090 0.126 0.225 0.255 Condition 40.066 0.072 0.084 0.112 0.180 0.203 Experimental control 0.067 0.1830.386 0.608 1.233 1.956

TABLE 5-2 Measurement value of Immediately after the 3-monthpreservation ALP activity Abs: 550 nm start of preservation 4° C. 25° C.32° C. 37° C. 42° C. Condition 1 0.288 0.291 0.287 0.280 0.229 0.140Condition 2 0.264 0.277 0.280 0.285 0.232 0.151 Condition 3 0.277 0.2660.265 0.274 0.227 0.136 Condition 4 0.271 0.288 0.275 0.289 0.265 0.189Experimental control 0.278 0.287 0.283 0.305 0.210 0.129

TABLE 5-3 Remaining % of Immediately after the 3-month preservation NADPamount start of preservation 4° C. 25° C. 32° C. 37° C. 42° C. Condition1 100 99 101 92 84 45 Condition 2 100 98 98 95 86 50 Condition 3 100 99100 97 88 45 Condition 4 100 100 101 101 90 59 Experimental control 10091 88 76 43 31

Example 6

Two different solutions were prepared by dissolving NADP (manufacturedby Oriental Yeast Co., Ltd.) as a coenzyme so as to be 1.25 mM in 20-mMsodium citrate buffers (pH 6.5) that contained 5% lactose described inExample 1 and 10 trehalose described in Example 2, respectively. Thesolutions were each dispensed into microtubes at an amount of 20 μl pertube and dried for 5 hours under the conditions at 30° C. with 15%humidity. After drying, the microtubes were preserved for 6 months at 4°C., 25° C., and 35° C. in an aluminum bag containing a desiccant andthen their backgrounds, ALP activities, and NADP-remaining amounts weredetermined, respectively.

As a result, as shown in Table 6-1 to Table 6-3, both 5% lactose and 10%trehalose showed good stabilities under any of conditions as comparedwith the experimental control.

TABLE 6-1 Background Immediately after the 6-month preservation Abs: 550nm start of preservation 4° C. 25° C. 32° C. 5% lactose 0.067 0.0790.097 0.141 10% trehalose 0.067 0.068 0.111 0.158 Experimental control0.067 0.183 0.386 0.608

TABLE 6-2 Measurement value of ALP activity Immediately after the6-month preservation Abs: 550 nm start of preservation 4° C. 25° C. 32°C. 5% lactose 0.276 0.303 0.281 0.257 10% trehalose 0.276 0.298 0.2570.248 Experimental control 0.278 0.238 0.262 0.292

TABLE 6-3 Remaining % of NADP Immediately after the 6-month preservationamount start of preservation 4° C. 25° C. 32° C. 5% lactose 100 102 9177 10% trehalose 100 105 95 95 Experimental control 100 99 92 65

Example 7

For one of two different anti-A influenza virus monoclonal antibodies(manufactured by Fitzgerald Co., Ltd.), 1 μl of an antibody solution (2mg/ml) was spotted on a nitrocellulose membrane (manufactured by WhatmanCo., Ltd.) of 25 mm×30 mm and then dried at 42° C. for 30 minutes. Thedried nitrocellulose membrane was then mounted on a water-absorbing pad.Subsequently, a top cover was closed so that a previously-openedobservation window in the cover corresponded to the spotted portion. Awater-soluble filter KC40 (manufactured by Aicello chemical Co., Ltd.)made of polyvinyl alcohol as shown in FIG. 1 was attached on the lowerpart of a enzyme substrate solution adding vessel and an enzymesubstrate filling port was formed in the opening of a top cover of asimplified immunoassay device, thereby completing a simplifiedimmunoassay device.

The other kind of the anti-A influenza virus monoclonal antibody inamount of 0.15 mg was labeled with a commercially available alkalinephosphatase labeling kit, thereby obtaining the labeled anti-A influenzavirus monoclonal antibody. This labeled antibody was diluted 4,000-foldinto a Tris-HCl buffer (pH 7.5) containing 2% BSA to prepare anenzyme-labeled antibody dilution.

A simulated sample was obtained by diluting 0.75 mg/ml of A-typeinfluenza virus antigen H3N2 (manufactured by HyTest Co., Ltd.) so as tobe 0.1 μg/ml with PBS containing 0.2% BSA and 0.1% Triton X-100 andsoaking 50 μl of the dilution into a swab. An experimental control usedwas a swab soaked with 50 μl of a dilute solution.

The swab well-soaked with the influenza antigen was placed in 0.2 ml ofthe enzyme-labeled antigen dilution, and the dilution was thensufficiently stirred, followed by adding the total amount thereof to thepreviously-formed observation window of the simple immunoassay device.Promptly without any interval, 500 μl of the enzyme-cycling reactionsolution of Example 1 was added to the enzyme substrate solution addingvessel. After 10 minutes from the addition, the presence or absence of ablue pigment on the nitrocellulose membrane was visually observed.

Results

Visual observation H3N2 antigen added + Experimental control −

As described above, it was judged positive only when the influenzaantigen was added, so a simple test using the simplified immunoassaydevice shown in FIG. 1 was able to be demonstrated.

Example 8

According to Example 6, the anti-A influenza virus monoclonal antibodywas immobilized on a nitrocellulose membrane and covered with a topcover. As a enzyme substrate solution adding vessel, a simplifiedimmunoassay device was formed such that a filter, which was soaked witha 40% sucrose solution and then dried, was attached on the upstreamportion of a siphon structure shown in FIG. 2 and an enzyme substratefilling port was formed in the opening of the top cover of thesimplified immunoassay device. The subsequent procedures were carriedout in a manner similar to Example 6. The presence or absence of a bluepigment on the nitrocellulose membrane was visually observed.

Results

Visual observation H3N2 antigen added + Experimental control −

As described above, it was judged positive only when the influenzaantigen was added, so a simple test using the simplified immunoassaydevice shown in FIG. 2 was able to be demonstrated.

INDUSTRIAL APPLICABILITY

The present invention provides a stabilization method for aphosphorylated coenzyme particularly at around room temperature, whichcan be preferably used in the fields of clinical diagnosis, foodevaluation, an examination for biogenic substances, and so on.

1. A stabilization method for a phosphorylated coenzyme, comprisingallowing at least a substance for suppressing dephosphorylation reactionof a phosphorylated coenzyme to coexist with a phosphorylated coenzyme.2. A stabilization method for a phosphorylated coenzyme in a dry state,comprising: allowing at least a substance for suppressingdephosphorylation reaction of a phosphorylated coenzyme to coexist witha phosphorylated coenzyme in a solution; and drying the solution.
 3. Thestabilization method according to claim 1, wherein the substance forsuppressing dephosphorylation reaction is a sugar and/or a sugaralcohol.
 4. The stabilization method according to claim 1, wherein thesubstance for suppressing dephosphorylation reaction is a polysaccharidecontaining glucose as a constituent.
 5. The stabilization methodaccording to claim 4, wherein the substance for suppressingdephosphorylation reaction is a disaccharide containing glucose as aconstituent.
 6. The stabilization method according to claim 5, whereinthe substance for suppressing dephosphorylation reaction is one or acombination of two or more selected from the group consisting oflactose, trehalose, maltose, and sucrose.
 7. The stabilization methodaccording to claim 2, wherein the drying is performed by air-drying. 8.The stabilization method according to claim 1, wherein thephosphorylated coenzyme is nicotinamide adenine dinucleotide phosphate(NADP).
 9. The method according to claim 1, wherein stabilization occursfor a prolonged period.
 10. A method for stabilizing a phosphorylatedcoenzyme, comprising allowing trehalose and at least one of lactose,maltose, and sucrose to coexist with the phosphorylated coenzyme. 11.The method according to claim 10, comprising allowing trehalose and atleast one of lactose, maltose, and sucrose to coexist with aphosphorylated coenzyme in a solution; and further comprising drying thesolution.